Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum

Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite–host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction–oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54-attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- a...